Mitochondrial Membrane Potential Analysis by Flow Cytometry

The mitochondrial membrane potential was determined using the MitoProbe JC‑1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole carbocyanide iodide) assay kit (Life Technologies, Carlsbad, CA). JC‑1 aggregates in intact mitochondria and emits red fluorescence. When the mitochondrial membrane potential is disrupted, JC‑1 remains in the cytoplasm as a monomer and emits green fluorescence. Primary preadipocytes that expanded from the stromal vascular fraction of BAT were treated with Pi (0 or 4 mM) for 16 hours in the presence of 0.5% FCS, and collected after trypsinization. A total of 1×10⁶ cells were incubated with 5 μg/ml of JC‑1 at 37°C for 20 minutes, and 10,000 cells were subjected to a flow cytometric analysis on a FACSCalibur flow cytometer. The percentage of JC‑1 monomers (green fluorescence) was determined. In order to confirm the sensitivity of JC‑1 to alterations in the mitochondrial membrane potential, 50 μM of carbonyl cyanide m‑chlorophenyl hydrazone was used as a chemical uncoupler.